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. 1994 Sep;3(9):1374–1382. doi: 10.1002/pro.5560030903

Human immunodeficiency virus-1 reverse transcriptase heterodimer stability.

J Lebowitz 1, S Kar 1, E Braswell 1, S McPherson 1, D L Richard 1
PMCID: PMC2142949  PMID: 7530541

Abstract

Structural and biochemical evidence strongly supports a heterodimeric (p66p51) active form for human immunodeficiency virus-1 reverse transcriptase (RT). Heterodimer stability was examined by sedimentation analysis as a function of temperature and ionic strength. Using NONLIN regression software, monomer-dimer-trimer and monomer-dimer-tetramer association models gave the best fit to the analytical ultracentrifuge sedimentation equilibrium data. The heterodimer is the predominant form of RT at 5 degrees C, with a dimerization Ka value of 5.2 x 10(5) M-1 for both models. Ka values of 2.1 x 10(5) and 3.8 x 10(5) M-1 were obtained for the respective association models at 20 degrees C. RT in 50 and 100 mM Tris, pH 7.0, completely dissociates at 37 degrees C and behaves as an ideal monomeric species. The dissociation of RT as a function of increasing temperature was also observed by measuring the decrease in sedimentation velocity (sw,20). If the stabilization of the heterodimer was due primarily to hydrophobic interactions we would anticipate an increase in the association from 21 degrees C to 37 degrees C. The opposite temperature dependence for the association of RT suggests that electrostatic and hydrogen bond interactions play an important role in stabilizing heterodimers. To examine the effect of ionic strength on p66p51 association we determined the changes in sw,20 as a function of NaCl concentration. There is a sharp decrease in sw,20 between 0.10 and 0.5 M NaCl, leading to apparent complete dissociation. The above results support a major role for electrostatic interactions in the stabilization of the RT heterodimer.

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Selected References

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  1. Babé L. M., Rosé J., Craik C. S. Synthetic "interface" peptides alter dimeric assembly of the HIV 1 and 2 proteases. Protein Sci. 1992 Oct;1(10):1244–1253. doi: 10.1002/pro.5560011003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beard W. A., Wilson S. H. Kinetic analysis of template.primer interactions with recombinant forms of HIV-1 reverse transcriptase. Biochemistry. 1993 Sep 21;32(37):9745–9753. doi: 10.1021/bi00088a029. [DOI] [PubMed] [Google Scholar]
  3. Becerra S. P., Kumar A., Lewis M. S., Widen S. G., Abbotts J., Karawya E. M., Hughes S. H., Shiloach J., Wilson S. H., Lewis M. S. Protein-protein interactions of HIV-1 reverse transcriptase: implication of central and C-terminal regions in subunit binding. Biochemistry. 1991 Dec 17;30(50):11707–11719. doi: 10.1021/bi00114a015. [DOI] [PubMed] [Google Scholar]
  4. Clark P. K., Ferris A. L., Miller D. A., Hizi A., Kim K. W., Deringer-Boyer S. M., Mellini M. L., Clark A. D., Jr, Arnold G. F., Lebherz W. B., 3rd HIV-1 reverse transcriptase purified from a recombinant strain of Escherichia coli. AIDS Res Hum Retroviruses. 1990 Jun;6(6):753–764. doi: 10.1089/aid.1990.6.753. [DOI] [PubMed] [Google Scholar]
  5. Divita G., Müller B., Immendörfer U., Gautel M., Rittinger K., Restle T., Goody R. S. Kinetics of interaction of HIV reverse transcriptase with primer/template. Biochemistry. 1993 Aug 10;32(31):7966–7971. doi: 10.1021/bi00082a018. [DOI] [PubMed] [Google Scholar]
  6. Divita G., Restle T., Goody R. S., Chermann J. C., Baillon J. G. Inhibition of human immunodeficiency virus type 1 reverse transcriptase dimerization using synthetic peptides derived from the connection domain. J Biol Chem. 1994 May 6;269(18):13080–13083. [PubMed] [Google Scholar]
  7. ENGLARD S., SINGER T. P. Physicochemical studies on beta-amylase. J Biol Chem. 1950 Nov;187(1):213–219. [PubMed] [Google Scholar]
  8. Hansen J., Schulze T., Mellert W., Moelling K. Identification and characterization of HIV-specific RNase H by monoclonal antibody. EMBO J. 1988 Jan;7(1):239–243. doi: 10.1002/j.1460-2075.1988.tb02805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hsieh J. C., Zinnen S., Modrich P. Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase. J Biol Chem. 1993 Nov 25;268(33):24607–24613. [PubMed] [Google Scholar]
  10. Huber H. E., McCoy J. M., Seehra J. S., Richardson C. C. Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching. J Biol Chem. 1989 Mar 15;264(8):4669–4678. [PubMed] [Google Scholar]
  11. Jacobo-Molina A., Ding J., Nanni R. G., Clark A. D., Jr, Lu X., Tantillo C., Williams R. L., Kamer G., Ferris A. L., Clark P. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6320–6324. doi: 10.1073/pnas.90.13.6320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnson M. L., Correia J. J., Yphantis D. A., Halvorson H. R. Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques. Biophys J. 1981 Dec;36(3):575–588. doi: 10.1016/S0006-3495(81)84753-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kati W. M., Johnson K. A., Jerva L. F., Anderson K. S. Mechanism and fidelity of HIV reverse transcriptase. J Biol Chem. 1992 Dec 25;267(36):25988–25997. [PubMed] [Google Scholar]
  14. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Larder B., Purifoy D., Powell K., Darby G. AIDS virus reverse transcriptase defined by high level expression in Escherichia coli. EMBO J. 1987 Oct;6(10):3133–3137. doi: 10.1002/j.1460-2075.1987.tb02623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lightfoote M. M., Coligan J. E., Folks T. M., Fauci A. S., Martin M. A., Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol. 1986 Nov;60(2):771–775. doi: 10.1128/jvi.60.2.771-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lowe D. M., Aitken A., Bradley C., Darby G. K., Larder B. A., Powell K. L., Purifoy D. J., Tisdale M., Stammers D. K. HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis. Biochemistry. 1988 Dec 13;27(25):8884–8889. doi: 10.1021/bi00425a002. [DOI] [PubMed] [Google Scholar]
  19. Reardon J. E. Human immunodeficiency virus reverse transcriptase: steady-state and pre-steady-state kinetics of nucleotide incorporation. Biochemistry. 1992 May 12;31(18):4473–4479. doi: 10.1021/bi00133a013. [DOI] [PubMed] [Google Scholar]
  20. Restle T., Müller B., Goody R. S. Dimerization of human immunodeficiency virus type 1 reverse transcriptase. A target for chemotherapeutic intervention. J Biol Chem. 1990 Jun 5;265(16):8986–8988. [PubMed] [Google Scholar]
  21. Restle T., Müller B., Goody R. S. RNase H activity of HIV reverse transcriptases is confined exclusively to the dimeric forms. FEBS Lett. 1992 Mar 23;300(1):97–100. doi: 10.1016/0014-5793(92)80172-d. [DOI] [PubMed] [Google Scholar]
  22. Teller D. C., Swanson E., de Haën C. The translational friction coefficient of proteins. Methods Enzymol. 1979;61:103–124. doi: 10.1016/0076-6879(79)61010-8. [DOI] [PubMed] [Google Scholar]
  23. Thimmig R. L., McHenry C. S. Human immunodeficiency virus reverse transcriptase. Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase. J Biol Chem. 1993 Aug 5;268(22):16528–16536. [PubMed] [Google Scholar]
  24. VINOGRAD J., BRUNER R., KENT R., WEIGLE J. Band-centrifugation of macromolecules and viruses in self-generating density gradients. Proc Natl Acad Sci U S A. 1963 Jun;49:902–910. doi: 10.1073/pnas.49.6.902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Whitcomb J. M., Hughes S. H. Retroviral reverse transcription and integration: progress and problems. Annu Rev Cell Biol. 1992;8:275–306. doi: 10.1146/annurev.cb.08.110192.001423. [DOI] [PubMed] [Google Scholar]
  26. Yphantis D. A. Pulsed laser interferometry in the ultracentrifuge. Methods Enzymol. 1979;61:3–12. doi: 10.1016/0076-6879(79)61003-0. [DOI] [PubMed] [Google Scholar]
  27. di Marzo Veronese F., Copeland T. D., DeVico A. L., Rahman R., Oroszlan S., Gallo R. C., Sarngadharan M. G. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science. 1986 Mar 14;231(4743):1289–1291. doi: 10.1126/science.2418504. [DOI] [PubMed] [Google Scholar]

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